The present invention concerns method and apparatus for detecting binding interactions in cells. The methods and apparatus are particularly suitable for the high through-put screening of cell arrays and combinatorial libraries.
A significant fraction of known cellular signaling proteins have been shown to translocate to or dissociate from the plasma membrane as part of their activation cycle. In particular, the recruitment of cytosolic proteins by activated receptors and plasma membrane signaling proteins is a general principle in receptor-mediated signal transduction (Pawson, T. (1995) Nature 373, 573-580; Ullrich, A. and Schlessinger, J. (1990) Cell 61, 203-212; Pfister, et al. (1985) Science 228, 891-893; Hunter, T. (1987) Cell 50, 823-829; Pawson, T. and Scott, J. D. (1997) Science 278, 2075-2080). Translocation is often transient with active signaling components dissociating from the plasma membrane and acting on cytosolic and nuclear targets. Recruitment processes are exemplified by the binding of cytosolic SH2-domain containing proteins to tyrosine phosphorylated plasma membrane receptors (Koch, et al. (1991) Science 252, 668-674; Kypta, et al. (1990) Cell 62, 481-492) and by the binding of cytosolic signaling enzymes to GTP bound small G-proteins at the plasma membrane (Moodie, et al. (1993) Science 260, 1658-1661; Stokoe, et al. Science 264, 1463-1467; Leevers, et al. (1994) Nature 369, 411-414). In addition to direct recruitment by protein-protein binding interactions, protein-lipid binding interactions are also important for translocation (Nishizuka, Y. (1992) Science 258, 607-614; Rameh, L. E. and Cantley, L. C. (1999) J. Biol. Chem. 274, 8347-8350). Lipid recruitment is exemplified by the translocation of PH-domain containing proteins in response to receptor-mediated production of plasma membrane phosphatidylinositol lipids (Ferguson, et al. (1995) Cell 83, 1037-1046), C1-domain containing proteins in response to plasma membrane diacylglycerol production, and C2-domain containing proteins in response to calcium mediated binding interactions with negatively charged lipids in the plasma membrane (Nishizuka, Y. (1992) Science 258, 607-614; Newton, A. C. (1995) Curr. Biol. 5, 973-976).
Why does translocation to the plasma membrane play such a ubiquitous role in signal transduction? First, most of the cellular interactions with the extracellular environment are mediated by receptors located in the plasma membrane. Activated receptors often serve as a scaffold for signaling proteins that have to be recruited for a particular signaling function. Second, plasma membrane translocation concentrates signaling proteins at the membrane and enhances the frequency of intermolecular collisions. Translocation then serves as an intermediate signaling step that enhances the effective on-rate for target binding or the Michaelis constant for enzyme action (Haugh, J. M. and Lauffenberger, D. A. (1997) Biophys. J. 72, 2014-2031).
Over the last few years, confocal imaging measurements were used to monitor the plasma membrane translocation of signaling proteins over time (Sakai, et al. (1997) J. Cell Biol. 139, 1465-1476; Venkateswarlu, et al. (1998) Curr. Biol. 8, 463-466; Barak, et al. (1997) J. Biol. Chem. 272, 27497-27500; Oancea, et al. (1997) J. Cell Biol. 140, 485-498; Stauffer, T. and Meyer, T. (1997) J. Cell Biol. 139, 1447-1454; Stauffer, et al. (1998) Curr. Biol. 8, 343-346; Kontos, et al. (1998) Mol. Cell Biol. 18: 4131-4140; Oancea, E. and Meyer, T. (1998) Cell 95, 307-318; Parent, et al. (1998) Cell 95, 81-91; Meili, et al. (1999) EMBO J. 18, 2092-2105; Watton, S. J. and Downward, J. (1999) Curr. Biol. 9, 433-436). Although successful for many proteins, this approach was limited to cell types where the confocal resolution was sufficient to separate the plasma membrane from the cytosol and where the translocation involved a significant fraction of the cytosolic protein. Nevertheless, these imaging studies showed that single cell time-course measurements of translocation events can give important insights into the activation mechanism of enzymes (Oancea, E. and Meyer, T. (1998) Cell 95, 307-318), into spatial gradients of second messengers (Parent, et al. (1998) Cell 95, 81-91; Meili, et al. (1999) EMBO J. 18, 2092-2105; Watton, S. J. and Downward, J. (1999) Curr. Biol. 9, 433-436) and into the single cell kinetics of specific signaling steps (Stauffer, T. and Meyer, T. (1997) J. Cell Biol. 139, 1447-1454; Oancea, E. and Meyer, T. (1998) Cell 95, 307-318).
Biomolecular or combinatorial arrays have provided a means for the high throughput screening of chemical libraries. See, e.g., U.S. Pat. No. 5,143,854. A variety of specific techniques for carrying out the automated screening of such arrays have been developed, including the evanescent scanning of a pixel array. See U.S. Pat. No. 5,633,724.
A disadvantage of combinatorial arrays is that they provide an in vitro rather than an in vivo assay. In vitro binding assays can seldom provide an accurate measure of how binding will actually occur in vivo, particularly for intracellular binding events, because the complexity of the intracellular environment is difficult to replicate outside of the cell. Of course, the ultimate application of many screening assays is to develop in vivo applications for the compounds being screened. Accordingly, there is a continued need for new in vivo screening techniques that can be readily adapted to automated or high throughput screening.
A first aspect of the present invention is an apparatus for screening for translocation of a first protein of interest in vivo in a cell. The apparatus comprises:
(a) a total internal reflection member having a surface portion. If desired, the surface portion can be divided into separate and discrete segments.
(b) A cell contacted to the surface portion by the plasma membrane of the cell, the protein having a fluorescent group conjugated thereto. If desired, different cells can be contacted to different ones of the separate and discrete segments.
(c) A light source operatively associated with the total internal reflection member and positioned for directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, with the evanescent field being weaker in a second portion of the cell, the fluorescent group emitting light when in the first portion of the cell and emitting less light when in the second portion of the cell (i.e., less light as compared to the amount emitted when the same fluorescent group is in the first portion of the cell).
(d) A light detector operatively associated with the total internal reflection member and configured to detect emitted light from the cell
The emission of more or less light from the cell indicates the translocation of the first protein between the first and second portions of the cell.
The cell or cells may further contain a second protein of interest located in either the first portion of the cell or the second portion of the cell, whereby the emission of more or less light from the cell indicates the presence or absence of specific binding between the first and second proteins of interest. When the second protein is located in the first portion of the cell, the emission of more light indicates the specific binding of the proteins of interest, and the emission of less light indicates the lack of such binding. When the second protein is located in the second portion of the cell, the emission of less light indicates the specific binding of the proteins of interest, and the emission of more light indicates the lack of such binding. First and second proteins of interest may be members of a specific binding pair. Either or both of the first and second proteins of interest may be expressed by a nucleic acid carried by the cell; either of the first and second proteins of interest may be a member of a library of compounds, with a different member of said library being expressed in cells of different segments, while the other protein of interest is the same in the cells of different segments, to provide a way to rapidly screen the library of compounds.
A second aspect of the present invention is a method of detecting translocation of a first protein of interest within a cell. The method comprises:
(a) providing a total internal reflection member having a surface portion, with a cell contacted to the surface portion by the plasma membrane of the cell;
(b) directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, the evanescent field being weaker in a second portion of the cell; wherein the protein of interest has a fluorescent group conjugated thereto; the fluorescent group emitting light when in the first portion of the cell and emitting less light when in the second portion of the cell; and then
(c) detecting emitted light from the fluorescent group, with the emission of more or less light from the fluorescent group indicating the translocation of the first protein of interest between the first and second portions of the cell.
The method may be used with a second protein of interest as described in connection with the apparatus above. An analysis of a test compound (e.g., a member of a library of compounds as described below) may be carried out by administering a test compound to the cell to determine whether or not said test compound disrupts the binding of said first and second proteins of interest. The analysis may be made a quantitative analysis by repeating steps (a) through (c) with different cells at different concentrations of said test compound. The degree of binding or disruption of binding may then be determined at different concentrations of the test compound.
The methods and apparatus of the invention can be used on individual cells or for screening multiple cell populations, or libraries of cells or libraries of compounds, as described in greater detail below.
A further aspect of the present invention is a method of screening binding between a first protein of interest and a library of second proteins of interest within a plurality of cells. The method comprises:
(a) providing a total internal reflection member having a surface portion, the surface portion having a plurality of separate and discrete segments, with a cell contacted to each of the surface portion segments by the plasma membrane of the cells;
(b) directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, the evanescent field being weaker in a second portion of the cell; wherein one of the proteins of interest has a fluorescent group conjugated thereto, and the other of the proteins of interest is located in either the first portion of the cell or the second portion of the cell; and wherein one of the proteins of interest is the same in each of the cells; and the other of the proteins of interest is a different member of the library in cells contacted to different segments; with the fluorescent group emitting light when in the first portion of each of the cells and emitting less light when in the second portion of each of the cells; and then
(c) detecting emitted light from each of the segments, with the presence or absence of emitted light indicating the presence or absence of specific binding between the proteins of interest in the cell in each of the segments.
A further aspect of the present invention is a method of screening a library of compounds for the ability to disrupt binding between first and second proteins of interest. The method comprises:
(a) providing a total internal reflection member having a surface portion, the surface portion having a plurality of separate and discrete segments, with a cell contacted to each of the surface portion segments by the plasma membrane thereof;
(b) directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, the evanescent field being weaker in a second portion of the cell;
wherein one of the proteins of interest has a fluorescent group conjugated thereto, and the other of the proteins of interest is located in either the first portion of the cell or the second portion of the cell;
the fluorescent group emitting light when in the first portion of each of the cells and emitting less light when in the second portion of each of the cells;
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(c) administering a different member of the library of compounds to each of the separate and discrete segments (e.g., by contacting a different compound to the cells, or by expressing a different compound from a different nucleic acid in each of said cells); and then
(d) detecting emitted light from the fluorescent group in the cells from each of the separate and discrete segment.
The presence or absence of emitted light from the fluorescent group indicates the disruption or lack of disruption of specific binding between the proteins of interest by the member of the library administered to the segment.
When screening libraries, the screening steps may be repeated with different members of the library until sufficient members of the library have been screened. It will also be appreciated that each cell may contain or be administered a sub-population or subpool of the library, and that where a population or subpopulation is found to contain a compound having desired properties, the screening step may be repeated with additional subpopulations containing the desired compound until the population has been reduced to one or a sufficiently small number to permit identification of the compound desired.
A further aspect of the present invention is an apparatus for screening for translocation of a first protein of interest in vivo in a cell. The apparatus comprises:
(a) a thin unitary total internal reflection member having a surface portion. If desired, the surface portion can be divided into separate and discrete segments. The total internal reflection member is preferably a single thin member which can be conveniently formed from a microscope slide coverslip, although other embodiments are also contemplated.
(b) A cell (typically a plurality of cells) contacted to the surface portion by the plasma membrane of the cell, the protein having a fluorescent group conjugated thereto. If desired, different cells can be contacted to different ones of the separate and discrete segments.
(c) A light source operatively associated with the total internal reflection member and positioned for directing a source light into the member to produce an evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, with the evanescent field being weaker in a second portion of the cell, the fluorescent group emitting light when in the first portion of the cell and emitting less light when in the second portion of the cell (i.e., less light as compared to the amount emitted when the same fluorescent group is in the first portion of the cell).
(d) coupling means such as a cylindrical lens or lenses, or a focused laser used in combination with a one-dimensional scanning mirror, etc., for coupling the light source to the thin unitary total internal reflection member, preferably thereby providing wide-field illumination of the surface portion of the total internal reflection member.
(e) A light detector operatively associated with the total internal reflection member and configured to detect emitted light from the cell
The emission of more or less light from the cell indicates the translocation of the first protein between the first and second portions of the cell.
The cell or cells may further contain a second protein of interest located in either the first portion of the cell or the second portion of the cell, whereby the emission of more or less light from the cell indicates the presence or absence of specific binding between the first and second proteins of interest. When the second protein is located in the first portion of the cell, the emission of more light indicates the specific binding of the proteins of interest, and the emission of less light indicates the lack of such binding. When the second protein is located in the second portion of the cell, the emission of less light indicates the specific binding of the proteins of interest, and the emission of more light indicates the lack of such binding. First and second proteins of interest may be members of a specific binding pair. Either or both of the first and second proteins of interest may be expressed by a nucleic acid carried by the cell; either of the first and second proteins of interest may be a member of a library of compounds, with a different member of said library being expressed in cells of different segments, while the other protein of interest is the same in the cells of different segments, to provide a way to rapidly screen the library of compounds.
A further aspect of the present invention is a method of detecting translocation of a first protein of interest within a cell. The method comprises:
(a) providing a thin unitary total internal reflection member having a surface portion, with a cell contacted to the surface portion by the plasma membrane of the cell;
(b) directing a source light into the member through a coupling means to produce a wide-field evanescent field adjacent the surface portion, with the evanescent field extending into a first portion of the cell adjacent the plasma membrane, the evanescent field being weaker in a second portion of the cell; wherein the protein of interest has a fluorescent group conjugated thereto; the fluorescent group emitting light when in the first portion of the cell and emitting less light when in the second portion of the cell; and then
(c) detecting emitted light from the fluorescent group, with the emission of more or less light from the fluorescent group indicating the translocation of the first protein of interest between the first and second portions of the cell.
The method may be used with a second protein of interest as described in connection with the apparatus above. An analysis of a test compound (e.g., a member of a library of compounds as described below) may be carried out by administering a test compound to the cell to determine whether or not said test compound disrupts the binding of said first and second proteins of interest. The analysis may be made a quantitative analysis by repeating steps (a) through (c) with different cells at different concentrations of said test compound. The degree of binding or disruption of binding may then be determined at different concentrations of the test compound.
The methods and apparatus of the invention can be used on individual cells or for screening multiple cell populations, or libraries of cells or libraries of compounds, as described in greater detail below.
The present invention is explained in greater detail in the drawings herein and the specification set forth below.